Genome-wide gene expression analysis in cancer cells reveals 3D growth to affect ECM and processes associated with cell adhesion but not DNA repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact.     

Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15–19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.Since its first demonstration in (live) cell imaging (1), stimulated emission depletion (STED) fluorescence microscopy has been realized in many variants. Particularly, the key phenomenon employed in this method, namely switching fluorophores transiently off by stimulated emission, has been accomplished with laser pulses varying from picoseconds to nanoseconds in duration, and from kHz to MHz in repetition rate. Because continuous-wave beams are suitable as well (2), STED microscopy has been implemented with rather different laser systems, ranging from model-locked femtosecond to continuous-wave laser diodes (3,4). Although it underscores the versatility of STED to modulate the fluorescence capability of a fluorophore, this wide range of options may confuse adopters when balancing simplicity, applicability, and resolution gain. The situation is exacerbated when implementing pairs of excitation and STED beams for dual-color colocalization studies (5,6).Here we report on a simple arrangement providing dual-color STED nanoscopy (Fig. 1) and molecular diffusion quantification down to ∼20 nm in (living) cells. The presented dual-channel STED microscope utilizes a single fiber laser providing a 20-MHz train of 775 nm wavelength pulses of 1.2-ns duration. This compact laser source enables STED on fluorophores emitting in the orange to red range. Specifically, we applied this laser on the orange dyes Atto590 and Atto594 (excitation: 595 nm; detection: 620 ± 20 nm), and the red dyes KK114 and Abberior Star635P (excitation: 640 nm; detection: 670 ± 20 nm). Although the spectra of the dyes are partially overlapping, the individual color channels can be separated without data processing (see Fig. S1 and Fig. S2 in the Supporting Material). Both channels are recorded simultaneously within 50 ns, using temporally interleaved pulsed excitation in combination with time-gated detection (5,7,8).Open in a separate windowFigure 1Fluorescence nanoscopy of protein complexes with a compact near-infrared nanosecond-pulsed STED microscope. (A) STED reveals immunolabeled subunits in amphibian NPC; raw data smoothed with a Gaussian filter extending over 14 nm in FWHM. The diameter of the octameric gp210 ring is established as ∼160 nm. Scale bar, 500 nm. (B) Individual NPC image showing eight antibody-labeled gp210 homodimers as 20–40 nm sized units and a 80 nm-sized localization of the subunits in the central channel.Because in STED microscopy, the STED doughnuts firmly determine the position of the fluorescently active molecules, the use of a single doughnut for both fluorophores guarantees that the two color channels are almost perfectly coaligned. The use of the doughnut even counteracts misalignments of the confocal excitation and detection channels (Fig. 2, and see Fig. S3), making STED microscopy particularly powerful for colocalization measurements.Open in a separate windowFigure 2Determination of the colocalization accuracy. Xenopus A6 cells, labeled with an antiserum against multiple NUP subunits in the central NPC channel and two secondary antibodies decorated with the fluorophores Abberior STAR635P and Atto594 were imaged by STED microscopy. (A) Upon overlaying both channels, a high degree of colocalization is directly visible. Scale bar, 200 nm. (B) Quantification of the colocalization by cross correlation of much larger images (see Fig. S3). The correlation is maximal for zero displacement of the images, proving colocalization. (C) Confocal image of monocolored fluorescent beads taken with improperly coaligned excitation beams (left). Improper coalignment spoils the colocalization accuracy in confocal imaging; the two channels should be perfectly coaligned, but they show a false offset as indicated by the color difference. The offset is quantified by the cross correlation of the two channels (right). (D) The STED image of the same beads (left) not only shows 10-fold improved resolution over the confocal image in panel C, but also improved colocalization, again quantified by cross correlation (right). Thus, by predetermining the position of emission, the STED doughnut counteracts errors induced by imperfect coalignment of the two confocal color channels (for details, see Fig. S3). Scale bars = 100 nm.The cross section for stimulated emission is lower at 775 nm as compared to that found at somewhat shorter wavelengths (5), yet STED pulse energies of ∼7 nJ in the focus are sufficient to yield a resolution of ∼30 nm and ∼20 nm in the orange and red channels, respectively (see Fig. S4). In addition, due to the lower peak intensity, the 1.2 ns pulses are likely to induce less nonlinear absorption and hence less photostress as compared to their more commonly used <0.2 ns counterparts (8,9). On the other hand, the pulses are only 2–4 times shorter than the typical lifetime of the excited state, which lessens their STED efficiency. This slight reduction is neutralized here by detecting photons emitted ∼1 ns after excitation (5,7,8).The potential of this straightforward implementation of STED microscopy is evident when imaging immunolabeled nuclear pore complexes (NPCs) of cultured Xenopus cells. Contrary to the confocal recording, STED microscopy reveals subunits of this protein complex, specifically the typical eightfold symmetry of its peripheral transmembrane protein gp210, along with a set of proteins in the central pore channel (Fig. 1, and see Fig. S5 and Fig. S6). Unlike in stochastic superresolution imaging of gp210 (10), the color channels are inherently coaligned and simultaneously recorded simply by executing a single scan. Apart from a weak smoothing and background subtraction applied to enhance image contrast, the images are raw.Because fluorescence off-switching by STED is an instant process, STED microscopy can be employed to study fast spatial translocations, such as the diffusion of molecules on the nanoscale (3). To benchmark the performance of our setup, we analyzed the diffusion of a fluorescent glycerophospholipid analog (11) by fluorescence correlation spectroscopy (FCS) in membranes of living mammalian PtK2-cells (Fig. 3). STED allowed us to reduce the diameter of the probed area from the 250 nm-sized diffraction limit down to 19 nm (FWHM), representing σ = 8 nm in standard deviation of a Gaussian fit. The attained subdiffraction area is 2.5 times smaller as compared to what has been reported in living cells to date (4). In model membranes, the smallest diameter was 15 nm (σ = 6.4 nm).Open in a separate windowFigure 3Nanoscale molecular diffusion analyzed by STED FCS. (A) For moderate and larger STED beam power P_STED, the resolution scales inversely with its square-root, attaining 15 nm in FWHM of the distribution of fluorescence emission in space, describing the measurement area. Note the relatively small threshold power P_S = 1.4 mW, which implies that a large resolution gain is already attained for P_STED < 100 mW. (Inset) The resolution was determined by measuring the transit time of a fluorescent phospholipid-analog (DSPE-PEG-KK114) in a lipid model membrane through the detection area by FCS. (B) In living mammalian Ptk2-cells, the transit time of the lipid analog scales linearly with the detection area, revealing a diffusion constant D_lat = 0.33 μm²/s, and showing that this lipid analog diffuses largely freely in the plasma membrane down to <20 nm scales.In both measurements, the molecular transit time depends linearly on the probed area, indicating that the labeled lipid molecules diffuse essentially freely down to spatial scales of 20 nm. Accordingly, the anomaly exponent α was close to 1 with values of α > 0.85, showing only minor deviations from free diffusion (see Fig. S7). Because the diameter is inversely proportional to the square-root of the STED beam power, the resolution can be adapted to a particular application need (Fig. 3, A and B).In summary, our arrangement provides up-to-date STED microscopy resolution in offset-free colocalization recordings. The ready-to-use near-infrared laser pulses keep undesired single and multiphoton absorption low and leave the visible spectrum amenable for further studies.     

Phospholipases. II. Enzymatic hydrolysis of lecithin: Effects of structure,cholesterol content,and sonication

Summary Hydrolysis of unsonicated liposomes of egg lecithin catalyzed by several phospholipases is markedly activated by addition ofn-alkanols [Jain & Cordes,J. Membrane Biol. 14:101 (1973)]. Further pursuit of these systems has established that several factors, including higher temperatures, increasing unsaturation of fatty acyl chains of the substrate, incorporation of cholesterol into the liposomes, and sonication, reduce the concentration ofn-hexanol required to elicit maximal activation for enzymatic hydrolysis. Moreover, sonication or incorporation of cholesterol into lecithin liposomes reduces from C₈ to C₇ and C₆, respectively, the chain length of that alcohol eliciting maximal activation. These results are consistent with the hypothesis that sonication and increasing cholesterol content lead to liposomes which have a diminished thickness of the hydrocarbon region compared to that for unmodified liposomes derived from the same lecithin.     

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.     

Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage lambda

The gene regulatory circuitry of phage lambda is among the best-understood circuits. Much of the circuitry centres around the immunity region, which includes genes for two repressors, CI and Cro, and their cis-acting sites. Related phages, termed lambdoid phages, have different immunity regions, but similar regulatory circuitry and genome organization to that of lambda, and show a mosaic organization, arising by recombination between lambdoid phages. We sequenced the immunity regions of several wild phages with the immunity specificity of lambda, both to determine whether natural variation exists in regulation, and to analyse conservation and variability in a region rich in well-studied regulatory elements. CI, Cro and their cis-acting sites are almost identical to those in lambda, implying that regulatory mechanisms controlled by the immunity region are conserved. A segment adjacent to one of the operator regions is also conserved, and may be a novel regulatory element. In most isolates, different alleles of two regulatory proteins (N and CII) flank the immunity region; possibly the lysis-lysogeny decision is more variable among isolates. Extensive mosaicism was observed for several elements flanking the immunity region. Very short sequence elements or microhomologies were also identified. Our findings suggest mechanisms by which fine-scale mosaicism arises.     

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.     

Mark-resight estimates of seasonal variation in harbor seal abundance and site fidelity

Monitoring trends in abundance of pinnipeds typically involves counting seals at terrestrial haul-out sites during the breeding season. Counts of seals made at other times of the year are typically lower; however, it is often unknown whether this is because fewer animals are present or whether lower counts simply reflect a reduction in haul-out probability. Here we illustrate how photo-identification data from an individual-based study of harbor seals (Phoca vitulina) can be used to estimate seasonal variation in abundance and site fidelity. Monthly data collected over a two-year period were analyzed using a mark-recapture mark-resight model accounting for individuals transitioning between observable and unobservable states. Levels of site fidelity were high throughout the year and abundance estimates showed no seasonal pattern. This suggests that individual seals used haul-out sites to the same extent throughout the year, and that peaks in counts during the breeding season are a result of seasonal changes in haul-out probability. The results of this study have implications for understanding population sub-structuring, gene flow and disease spread.     

Trail‐sharing among tropical ants: interspecific use of trail pheromones?

1. Trail‐sharing between different ant species is rare and restricted to a small number of species pairs. Its underlying mechanisms are largely unknown. For trail‐sharing to occur, two factors are required: (i) one or both species must recognise the other species or its pheromone trails and (ii) both species must tolerate each other to a certain extent to allow joint use of the trail. A species that follows another's trails can efficiently exploit the other's information on food sources contained in the pheromone trails. Hence, food competition and thus aggressive interactions between a species following another's trail and the species being followed, seem likely. 2. In the present study, we investigated interspecific trail following and interspecific aggression in trail sharing associations (i) among Polyrhachis ypsilon, Camponotus saundersi, and Dolichoderus cuspidatus, and (ii) among Camponotus rufifemur and Crematogaster modiglianii. We tested whether trail‐sharing species follow each other's pheromone trails, and whether the ants tolerated or attacked their trail‐sharing partners. In both associations, we confronted workers with pheromone trails of their associated species, and, for the former association, measured interspecific aggression among the trail‐sharing species. 3. In our assays, D. cuspidatus and C. rufifemur regularly followed heterospecific pheromone trails of P. ypsilon and C. modiglianii, respectively. However, only few workers of the remaining species followed heterospecific pheromone trails. Thus, shared trails of P. ypsilon and C. saundersi cannot be explained by interspecific trail‐following. 4. Interspecific aggression among P. ypsilon, C. saundersi, and D. cuspidatus was strongly asymmetric, C. saundersi being submissive to the other two. All three species differentiated between heterospecific workers from the same or another site, suggesting habituation to the respective trail‐sharing partners. We therefore hypothesise that differential tolerance by dominant ant species may be mediated by selective habituation towards submissive species and this way determines the assembly of trail‐sharing associations.